The cloning of the gene mutated in patients with cystic fibrosis (CF) has made possible interpretation of the deduced primary structure of the gene product, CFTR. In the context of what was known of an epithelial Cl.sup.- permeability defect in CF, this lead to the original suggestion that the gene coded for either a Cl.sup.- channel itself or a regulator of a separate Cl.sup.- channel (Riordan et al, 1989). The introduction of expressible CFTR cDNAs into cells bearing CF-causing mutations in the gene (Rich et al, 1990; Druman et al, 1990), or into cells in which CFTR is not normally expressed (Anderson et al, 1991a; Kartner et al, 1991; Rich et al, 1991; Bear et al, 1991; Dalemans et al, 1991; Drumm et al, 1991) resulted in the appearance of a Cl.sup.- conductance regulated by cyclic AMP and similar to that seen in several normal epithelial cell types (Gray et al, 1989; Tabcharani et al, 1990). A low conductance ohmic Cl.sup.- channel activated by protein kinase A (PKA)--catalysed phosphorylation and inactivated by dephosphorylation was shown to underlie this conductance pathway (Tabcharani et al, 1991; Berger et al, 1991). Although these findings cannot distinguish between the CFTR protein constituting the conductance pathway itself, or its being a phosphorylation-activated regulator, changes in ion selectivity on mutation of amino acids with charged side chains in the proposed transmembrane sequences (viz. K95 in TM1 and K335 in TM6; Anderson et ai, 1991b) tend to support the former possibility.
Consistent with its proposed role as an ion channel, CFTR is a relatively non-abundant protein in the epithelial tissues in which it is endogenously expressed. We know of no tissue which provides an adequate source for purification. Similarly, it has not yet been possible to establish mammalian cell lines in which a very high level of heterologous expression of CFTR occurs (Cheng et al, 1990). This is believed to be at least partially due to a rather stringent control of CFTR biosynthesis which limits the amount of wild type protein which accumulates in cells (Gregory et al, 1991). This quality control is apparently more strictly enforced in the case of some mutant forms of CFTR, including the product of the most common mutation (F508), in which little or no mature protein is detectable and only small amounts of immature precursor is present, apparently in the endoplasmic reticulum (Cheng et al, 1990).
Until the work of the present inventors, no one had succeeded in isolating CFTR and purifying it to substantial homogeneity.